ab174443 Interferon-gamma (IFNG) Human SimpleStep ELISA Kit Instructions for Use For the quantitative measurement of IFN gamma in human cell culture supernatant, plasma and serum samples. This product is for research use only and is not intended for diagnostic use. Last Updated 26 April 2017
Table of Contents INTRODUCTION 1. BACKGROUND 2 2. ASSAY SUMMARY 3 GENERAL INFORMATION 3. 4. PRECAUTIONS STORAGE AND STABILITY 4 4 5. MATERIALS SUPPLIED 4 6. MATERIALS REQUIRED, NOT SUPPLIED 5 7. LIMITATIONS 5 8. TECHNICAL HINTS 5 ASSAY PREPARATION 9. REAGENT PREPARATION 7 10. STANDARD PREPARATION 8 11. SAMPLE PREPARATION 9 12. PLATE PREPARATION 10 ASSAY PROCEDURE 13. ASSAY PROCEDURE 11 DATA ANALYSIS 14. CALCULATIONS 13 15. TYPICAL DATA 14 16. TYPICAL SAMPLE VALUES 16 17. ASSAY SPECIFICITY 18 18. SPECIES REACTIVITY 20 RESOURCES 19. TROUBLESHOOTING 21 20. NOTES 22 1
INTRODUCTION 1. BACKGROUND Interferon-gamma (IFNG) in vitro SimpleStep ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for the quantitative measurement of Interferon-gamma protein in human cell culture supernatant, plasma and serum samples. The SimpleStep ELISA employs an affinity tag labeled capture antibody and a reporter conjugated detector antibody which immunocapture the sample analyte in solution. This entire complex (capture antibody/analyte/detector antibody) is in turn immobilized via immunoaffinity of an anti-tag antibody coating the well. To perform the assay, samples or standards are added to the wells, followed by the antibody mix. After incubation, the wells are washed to remove unbound material. TMB substrate is added and during incubation is catalyzed by HRP, generating blue coloration. This reaction is then stopped by addition of Stop Solution completing any color change from blue to yellow. Signal is generated proportionally to the amount of bound analyte and the intensity is measured at 450 nm. Optionally, instead of the endpoint reading, development of TMB can be recorded kinetically at 600 nm. IFN gamma is produced by lymphocytes activated by specific antigens or mitogens. IFN gamma, in addition to having antiviral activity, has important immunoregulatory functions. It is a potent activator of macrophages, it has antiproliferative effects on transformed cells and it can potentiate the antiviral and antitumor effects of the type I interferons. 2
INTRODUCTION 2. ASSAY SUMMARY Remove appropriate number of antibody coated well strips. Equilibrate all reagents to room temperature. Prepare all reagents, samples, and standards as instructed. Add standard or sample to appropriate wells. Add Antibody Cocktail to all wells. Incubate at room temperature. Aspirate and wash each well. Add TMB Substrate to each well and incubate. Add Stop Solution at a defined endpoint. Alternatively, record color development kinetically after TMB substrate addition. 3
GENERAL INFORMATION 3. PRECAUTIONS Please read these instructions carefully prior to beginning the assay. All kit components have been formulated and quality control tested to function successfully as a kit. Modifications to the kit components or procedures may result in loss of performance. 4. STORAGE AND STABILITY Store kit at 2-8ºC immediately upon receipt. Refer to list of materials supplied for storage conditions of individual components. Observe the storage conditions for individual prepared components in sections 9 & 10. 5. MATERIALS SUPPLIED 10X IFNG Capture Antibody 600 µl Storage Condition (Before Preparation) +2-8ºC 10X IFNG Detector Antibody 600 µl +2-8ºC IFNG Human Lyophilized Recombinant Protein 2 Vials +2-8ºC Antibody Diluent CPI 6 ml +2-8ºC 10X Wash Buffer PT 20 ml +2-8ºC TMB Substrate 12 ml +2-8ºC Stop Solution 12 ml +2-8ºC Sample Diluent NS 50 ml +2-8ºC Sample Diluent NBP Pre-Coated 96 Well Microplate (12 x 8 well strips) Plate Seal 20 ml +2-8ºC 96 Wells +2-8ºC 1 +2-8ºC Item Amount 4
GENERAL INFORMATION 6. MATERIALS REQUIRED, NOT SUPPLIED These materials are not included in the kit, but will be required to successfully utilize this assay: Microplate reader capable of measuring absorbance at 450 or 600 nm. Method for determining protein concentration (BCA assay recommended). Deionized water. Multi- and single-channel pipettes. Tubes for standard dilution. Plate shaker for all incubation steps. Optional: Phenylmethylsulfonyl Fluoride (PMSF) (or other protease inhibitors). 7. LIMITATIONS Assay kit intended for research use only. Not for use in diagnostic procedures. Do not mix or substitute reagents or materials from other kit lots or vendors. Kits are QC tested as a set of components and performance cannot be guaranteed if utilized separately or substituted. 8. TECHNICAL HINTS Samples generating values higher than the highest standard should be further diluted in the appropriate sample dilution buffers. Avoid foaming components. Avoid cross contamination of samples or reagents by changing tips between sample, standard and reagent additions. Ensure plates are properly sealed or covered during incubation steps. or bubbles when mixing or reconstituting 5
GENERAL INFORMATION Complete removal of all solutions and buffers during wash steps is necessary to minimize background. As a guide, typical ranges of sample concentration for commonly used sample types are shown below in Sample Preparation (section 11). All samples should be mixed thoroughly and gently. Avoid multiple freeze/thaw of samples. Incubate ELISA plates on a plate shaker during all incubation steps. When generating positive control samples, it is advisable to change pipette tips after each step. The provided Antibody Diluent and Sample Diluent contain protease inhibitor aprotinin. Additional protease inhibitors can be added if required. This kit is sold based on number of tests. A test simply refers to a single assay well. The number of wells that contain sample, control or standard will vary by product. Review the protocol completely to confirm this kit meets your requirements. Please contact our Technical Support staff with any questions. 6
ASSAY PREPARATION 9. REAGENT PREPARATION Equilibrate all reagents to room temperature (18-25 C) prior to use. The kit contains enough reagents for 96 wells. The sample volumes below are sufficient for 48 wells (6 x 8-well strips); adjust volumes as needed for the number of strips in your experiment. Prepare only as much reagent as is needed on the day of the experiment. Capture and Detector Antibodies have only been tested for stability in the provided 10X formulations. 9.1 1X Wash Buffer PT Prepare 1X Wash Buffer PT by diluting 10X Wash Buffer PT with deionized water. To make 50 ml 1X Wash Buffer PT combine 5 ml 10X Wash Buffer PT with 45 ml deionized water. Mix thoroughly and gently. 9.2 Antibody Cocktail Prepare Antibody Cocktail by diluting the capture and detector antibodies in Antibody Diluent CPI. To make 3 ml of the Antibody Cocktail combine 300 µl 10X Capture Antibody and 300 µl 10X Detector Antibody with 2.4 ml Antibody Diluent CPI. Mix thoroughly and gently. 7
ASSAY PREPARATION 10. STANDARD PREPARATION Prepare serially diluted standards immediately prior to use. Always prepare a fresh set of positive controls for every use. The following section describes the preparation of a standard curve for duplicate measurements (recommended). 10.1 For serum and plasma samples, reconstitute the IFNG protein standard sample by adding 200 µl Sample Diluent NBP. Note: Sample Diluent NBP should be warmed to 37 ºC for 10 minutes and mixed thoroughly, then equilibrated to room temperature before use. For cell culture supernatant samples, reconstitute the IFNG protein standard sample by adding 200 µl distilled water. Mix thoroughly and gently. Hold at room temperature for 10 minutes and mix gently. This is the 120 ng/ml Stock Standard Solution. 10.2 Label eight tubes with numbers 1 8. 10.3 For serum and plasma samples add 225 μl Sample Diluent NBP into tube number 1 and 150 μl into numbers 2-8. For cell culture supernatant samples add 225 μl Sample Diluent NS into tube number 1 and 150 μl into numbers 2-8. 10.4 Use the Stock Standard to prepare the following dilution series. Standard #8 contains no protein and is the Blank control 75 µl 150 µl 150 µl 150 µl 150 µl 150 µl 150 µl µ 120 ng/ml 30 ng/ml µ µ µ 7.5 3.75 15 1.875 ng/ml ng/ml ng/ml ng/ml µ µ 0 0.937 0.468 ng/ml ng/ml ng/ml 8
ASSAY PREPARATION 11. SAMPLE PREPARATION TYPICAL SAMPLE DYNAMIC RANGE Sample Type Range 48 hours PHA-stimulated PBMC supernatant 160 5X diluted 11.1 Plasma Collect plasma using citrate, EDTA or heparin. Centrifuge samples at 2,000 x g for 10 minutes. Neat plasma samples can be assayed without dilution. If needed, plasma samples should be diluted in the same negative matrix (negative plasma as appropriate) provided by a user. Heparin-plasma samples are not compatible with this assay kit because linearity of dilution cannot be guaranteed. 11.2 Serum Samples should be collected into a serum separator tube. After clot formation, centrifuge samples at 2,000 x g for 10 minutes and collect serum. Neat serum samples can be used without dilution. If needed, serum samples should be diluted in the same negative matrix (negative serum as appropriate) provided by a user. 11.3 Cell Culture Supernatants Centrifuge cell culture media at 2,000 x g for 10 minutes to remove debris. Collect supernatants, dilute them at least two fold in Sample Diluent NS and assay. 9
ASSAY PREPARATION 12. PLATE PREPARATION The 96 well plate strips included with this kit are supplied ready to use. It is not necessary to rinse the plate prior to adding reagents. Unused plate strips should be immediately returned to the foil pouch containing the desiccant pack, resealed and stored at 4 C. For each assay performed, a minimum of two wells must be used as the zero control. For statistical reasons, we recommend each sample should be assayed with a minimum of two replicates (duplicates). Differences in well absorbance or edge effects have not been observed with this assay. 10
ASSAY PROCEDURE 13. ASSAY PROCEDURE Equilibrate all materials and prepared reagents to room temperature prior to use. It is recommended to assay all standards, controls and samples in duplicate. 13.1 Prepare all reagents, working standards, and samples as directed in the previous sections. 13.2 Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, reseal and return to 4ºC storage. 13.3 Add 50 µl of all samples and standards to appropriate wells. 13.4 Add 50 µl of the Antibody Cocktail to each well. 13.5 Seal or cover plate and incubate for 1 hour at room temperature on a plate shaker set to 400 rpm. 13.6 Wash each well with 3 x 350 µl 1X Wash Buffer PT. Wash by aspirating or decanting from wells then dispensing 350 µl 1X Wash Buffer PT into each well. Complete removal of liquid at each step is essential for good performance. After the last wash invert the plate and blot it against clean paper towels to remove excess liquid. 13.7 Add 100 µl of TMB Substrate to each well and incubate for 10 minutes in the dark on a plate shaker set to 400 rpm. 13.8 Add 100 µl of Stop Solution to each well. Shake plate on a plate shaker for 1 minute to mix. Record the OD at 450 nm. This is an endpoint reading. Alternative to 13.7 13.8: Instead of the endpoint reading at 450 nm, record the development of TMB Substrate kinetically. Immediately after addition of TMB Development Solution begin recording the blue color development with elapsed time in the microplate reader prepared with the following settings: 11
ASSAY PROCEDURE Mode: Kinetic Wavelength: 600 nm Time: up to 15 min Interval: 20 sec - 1 min Shaking: Shake between readings Note that an endpoint reading can also be recorded at the completion of the kinetic read by adding 100 µl Stop Solution to each well and recording the OD at 450 nm. 13.9 Analyze the data as described below. 12
DATA ANALYSIS 14. CALCULATIONS 14.1 Calculate the average absorbance value for the blank control (zero) standards. Subtract the average blank control standard absorbance value from all other absorbance values. 14.2 Create a standard curve by plotting the average blank control subtracted absorbance value for each standard concentration (y-axis) against the target protein concentration (x-axis) of the standard. Use graphing software to draw the best smooth curve through these points to construct the standard curve. Note: Most microplate reader software or graphing software will plot these values and fit a curve to the data. A four parameter curve fit (4PL) is often the best choice; however, other algorithms (e.g. linear, semi-log, log/log, 4 parameter logistic) can also be tested to determine if it provides a better curve fit to the standard values. 14.3 Determine the concentration of the target protein in the sample by interpolating the blank control subtracted absorbance values against the standard curve. Multiply the resulting value by the appropriate sample dilution factor, if used, to obtain the concentration of target protein in the sample. 14.4 Samples generating absorbance values greater than that of the highest standard should be further diluted and reanalyzed. Similarly, samples which measure at an absorbance values less than that of the lowest standard should be retested in a less dilute form. 13
DATA ANALYSIS 15. TYPICAL DATA TYPICAL STANDARD CURVE Data provided for demonstration purposes only. A new standard curve must be generated for each assay performed. Standard Curve Measurements (for cell culture supernatant samples) Conc. O.D. 450 nm Mean (ng/ml) 1 2 O.D. 0 0.146 0.125 0.136 0.468 0.151 0.153 0.152 0.937 0.206 0.192 0.199 1.875 0.289 0.292 0.290 3.75 0.499 0.471 0.485 7.50 0.911 0.918 0.914 15 1.791 1.785 1.788 30 3.564 3.595 3.580 Figure 1. Example IFNG standard curve for cell culture supernatant samples measurements. The IFNG standard curve was prepared as described in Section 10. Raw data values are shown in the table. Background-subtracted data values (mean +/- SD) are graphed. 14
DATA ANALYSIS Conc. Standard Curve Measurements (for serum/plasma samples) O.D. 450 nm Mean (ng/ml) 1 2 O.D. 0 0.051 0.053 0.052 0.468 0.061 0.066 0.064 0.937 0.074 0.072 0.073 1.875 0.088 0.093 0.091 3.750 0.120 0.120 0.121 7.5 0.233 0.236 0.234 15 0.488 0.466 0.477 30 1.224 1.247 1.235 Figure 2. Example IFNG standard curve for serum/plasma samples measurements. The IFNG standard curve was prepared as described in Section 10. Raw data values are shown in the table. Background-subtracted data values (mean +/- SD) are graphed. 15
DATA ANALYSIS 16. TYPICAL SAMPLE VALUES SENSITIVITY The calculated minimal detectable (MDD) dose is 0.47 ng/ml. The MDD was determined by calculating the mean of zero standard replicates (n=25) and adding 2 standard deviations then extrapolating the corresponding concentrations. RECOVERY (Sample spiking in representative sample matrices) Sample Type 50% Media (10F RPMI) Neat Normal Human Serum Neat Normal Human Plasma-EDTA Neat Normal Human Plasma-citrate Neat Normal Human Plasma-heparin Average % Recovery 86 102 122 107 244 Range (%) 77-93 98-104 117-124 96-115 197-298 LINEARITY OF DILUTION Dilution Factor Undiluted 2 4 8 16 Dilution Factor Undiluted 2 4 8 16 32 PHA PBMC SN (fold of dilution) 5 10 20 40 80 Interpolated value (ng/ml) 22.87 11.74 6.56 3.33 1.70 Neat Human serum + IFNG (ng/ml) 30.000 15.000 7.500 3.750 1.875 0.938 Interpolated value (ng/ml) 27.17 13.76 6.51 3.47 2.37 0.89 % Expected Value 100 103 115 117 119 % Expected Value 100 101 96 102 139 105 16
DATA ANALYSIS Dilution Factor Undiluted 2 4 8 16 Dilution Factor Undiluted 2 4 8 16 32 Dilution Factor Undiluted 2 4 8 16 Neat Human plasma-edta + IFNG (ng/ml) 30.000 15.000 7.500 3.750 1.875 Interpolated value (ng/ml) 31.27 14.64 7.13 3.44 1.53 Neat Human plasma-citrate + IFNG (ng/ml) 30.000 15.000 7.500 3.750 1.875 0.938 Interpolated value (ng/ml) 28.05 13.70 7.20 3.87 2.01 0.85 Neat Human plasma-heparin + IFNG (ng/ml) 30.000 15.000 7.500 3.750 1.875 Interpolated value (ng/ml) 33.63 16.76 7.36 2.44 0.61 % Expected Value 100 94 91 88 78 % Expected Value 100 98 103 110 115 96 % Expected Value 100 100 88 58 29 PRECISION Mean coefficient of variations of interpolated values from 3 concentrations of PHA-stimulated PBMC supernatant within the working range of the assay. n= CV (%) IntraAssay 5 1.1 InterAssay 3 7.9 17
DATA ANALYSIS 17. ASSAY SPECIFICITY This assay recognizes both native and recombinant human IFNG protein. IFNG concentrations in all ten individual donor normal human serum samples measured less than the MDD, 0.7 ng/ml. Figure 3. Comparison of secreted IFNG in unstimulated and PHA-stimulated Human PBMC. PBMC were grown in the absence or presence of phytohemagglutinin (PHA) for 2 days. IFNG concentrations were measured in 12X and 6X diluted cell culture supernatants of the unstimulated PBMC and the stimulated PBMC, and media. Raw data values (mean +/-SD, n=3) are graphed. The dotted line represents zero sample background. 18
DATA ANALYSIS Figure 4. Interpolated concentrations of secreted IFNG in unstimulated and PHA-stimulated human PBMC. The concentrations of IFNG were interpolated from data values shown in Figure 3 using IFNG standard curve and corrected for sample dilution. The mean IFNG concentration was determined to be 1.8 ng/ml in unstimulated PBMC supernatants and 177.2 ng/ml in stimulated PBMC supernatants. 19
DATA ANALYSIS 18. SPECIES REACTIVITY This kit detects IFNG in human cell culture supernatant, plasma and serum samples only. Heparin-plasma samples are not compatible with this assay kit because linearity of dilution cannot be guaranteed. Please contact our Technical Support team for more information. 20
RESOURCES 19. TROUBLESHOOTING Problem Cause Solution Inaccurate Pipetting Check pipettes Improper standard dilution Prior to opening, briefly spin the stock standard tube and dissolve the powder thoroughly by gentle mixing Incubation times too brief Ensure sufficient incubation times; increase to 2 or 3 hour standard/sample incubation Inadequate reagent volumes or improper dilution Check pipettes and ensure correct preparation Incubation times with TMB too brief Ensure sufficient incubation time until blue color develops prior addition of Stop solution Plate is insufficiently washed Review manual for proper wash technique. If using a plate washer, check all ports for obstructions. Contaminated wash buffer Prepare fresh wash buffer Low sensitivity Improper storage of the ELISA kit Store your reconstituted standards at -80 C, all other assay components 4 C. Keep TMB substrate solution protected from light. Precipitate in Diluent Precipitation and/or coagulation of components within the Diluent. Precipitate can be removed by gently warming the Diluent to 37ºC. Poor standard curve Low Signal Large CV 21
RESOURCES 20. NOTES 22
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